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Forced expression of RNF36 induces cell apoptosis

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Abstract

RNF36 (ring finger protein 36; alias Trif), a member of the RING zinc finger protein family, is expressed in germ cells at round spermatid stages during spermatogenesis. RING finger proteins have been implicated in a variety of functions including oncogenesis, viral replication, and developmental processes. Since no germ cell line is presently available to study the function of RNF36, in this research, we expressed RNF36 truncated and full-length proteins in COS-7 and HEK-293 cell lines to study the effect of RNF36 in somatic cells. The full-length RNF36 protein in both cell lines showed a speckled pattern in the nucleus. Truncated RNF36-1 protein with its putative nuclear localization signal (NLS) remained within the nucleus but lost the speckled pattern. The promyelocytic leukemia (PML) protein, another RING finger protein, was previously identified as present in the nucleus with a speckled pattern. Double-staining and coimmunoprecipitation analyses suggested that RNF36 colocalizes and interacts with PML. In vitro phosphorylation analysis further suggested that RNF36 nuclear localization is under the control of phosphorylation, which might be mediated by p38. Treatment with the p38 inhibitor SB203580 resulted in the cytoplasmic translocation of RNF36. Overexpression of full-length RNF36 in cells induced about half of the transfected cells to undergo cell death. The results of DNA fragmentation assays, flow cytometry assay, and TUNEL staining suggest that the death of RNF36-transfected cells was caused by apoptosis. Following further characterization of the molecular mechanism of RNF36-induced apoptosis, we found that the expression of Bax, caspase-2, and receptor-interacting protein were elevated upon RNF36 induction in test cells. These results suggest that RNF36 may interact with PML and induce cell apoptosis. We suspect that RNF36 may play a role in germ cell homeostasis during spermatogenesis.

Introduction

We have recently isolated a testis RING finger gene, RNF36, which belongs to a RING-B-box-coiled-coil (RBCC) family [1]. The RING finger motif is a distinct zinc binding domain found in over 100 proteins, including protooncoproteins such as promyelocytic leukemia (PML) protein, the breast cancer gene product BRCA1, and cytoplasmic proteins, including peroxisomal proteins [2]. RINGs are involved in protein interactions but may not bind nucleic acids directly and are often involved in macromolecular assemblages [3]. RING finger proteins have been shown to play important roles in signal transduction, transcriptional regulation, ubiquination, and apoptosis [4], [5]. Members of the RBCC family are characterized by their possession of a tripatic motif consisting of a RING finger, one or two B boxes, and an α-helical coiled-coil domain [2], [6], [7]. These domains are involved in protein–protein interactions and allow RBCC family members to participate in various cellular processes depending on their subcellular localization [3]. It is well documented that PML suppresses cell growth [8], [9], [10] and acts as a tumor suppressor in vivo [3], [11]. PML associates with transcriptional coactivators and repressors supportive of a role for PML in the regulation of gene expression [12], [13]. PML also interacts with the transcriptional coactivator cAMP response element binding protein (CBP) and colocalizes in PML nuclear bodies (NB) [14], [15], [16], [17]. It has been shown that the acetylated or transcriptionally active forms of chromatin are also associated with PML NB [18]. A role for PML in transcriptional activation is supported by the finding that PML activates Fos-mediated transcription of AP-1[19]. PML also interacts with tumor suppressor p53 and GATA-2, activating transcription of their target genes [20], [21], [22]. PML, however, can also act as a transcriptional repressor; when fused to the DNA-binding domain of Ga14, PML represses Ga14-mediated transcription, possibly through a mechanism that recruits histone deacetylases to the target promoter [13], [23]. PML also inhibits Spl-mediated transactivation of the epidermal growth factor receptor promoter and the transcriptional activation mediated by Tax and Daxx [15], [24], [25]. PML has also been shown to interact with multiple corepressors and to mediate the transcriptional repressive function of Mad [26].

Apoptosis is essentially a physiological process that controls cell population during development and throughout an organism’s life at the appropriate time. Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing, and DNA fragmentation, which often depend on RNA and protein synthesis in the dying cell [27], [28]. Several genes have been shown to mediate or modulate apoptotic pathways, which are the Fas system, the tumor-suppressor gene p53, and genes of the Bcl-2 gene family [29], [30], [31]. Members of the Bcl-2 family of proteins have been reported to be involved in the regulation of apoptosis in various cell types and may either inhibit (Bcl-2, Bcl-xL, Mcl-1, and Bcl-w) or promote (Bax, Bcl-xs, Bak, and Bad) apoptosis [32], [33]. Bcl-2 family members interact with each other in regulating apoptosis. The gene products Bax and Bcl-2 are capable of forming homo- and heterodimers. When Bax is in excess and Bax homodimers dominate, cells are susceptible to apoptosis; Bcl-2 blocks apoptosis, presumably by forming inactive Bax/Bcl-2 heterodimers. Apoptotic induction could, therefore, depend on the Bax/Bcl-2 ratio [34]. A quantitative analysis within a hematopoietic cell line has indicated that, when half or more endogenous Bax is heterodimerized with Bcl-2, apoptosis is repressed [35], [36].

The RNF36 gene was identified as specifically expressed in round spermatids of mouse testis [1]. In the present report, we mapped the RNF36 gene to mouse chromosome 2F1. Subcellular location analysis found that RNF36 had a speckled nuclear pattern when transfected into somatic cells and the presence of RNF36 in the nuclear bodies is probably due to an interaction between PML and RNF36. To further characterize the function of RNF36, we overexpressed RNF36 cDNA in COS-7 and HEK-293 cells, resulting in massive cell apoptosis. The cell death induced by overexpression of RNF36 might be through a PML-mediated pathway. In this study, we provide evidence that RNF36 plays a specific role in apoptosis and suggest that RNF36 may play an important role in germ cell homeostasis during spermatogenesis.

Section snippets

Isolation of BAC clone and chromosomal localization of RNF36 gene

A RNF36 cDNA fragment (GenBank accession no. AF334958; bp 194–1694) was used as a probe to screen a mouse BAC library (Genome system). Four positive clones were isolated, one of which was used as a probe for fluorescence in situ hybridization (FISH) to identify the chromosomal location of RNF36. FISH was performed as described previously [37]. A BAC probe, labeled with digoxigenin dUTP by nick translation, was combined with sheared mouse DNA and hybridized to normal metaphase chromosomes

Chromosomal mapping of RNF36

To investigate whether the RNF36 gene is localized to a region known to be involved in reproduction, FISH was performed using a RNF36-containing BAC probe to map the chromosomal location of RNF36. The probe hybridized to the distal region of mouse chromosome 2 as revealed by DAPI staining (Fig. 1A). Using a specific marker, D2MIT354 hybridized to the centromeric region of chromosome 2 and cohybridized with RNF36 (Fig. 1B). Measurements of 10 specifically labeled chromosome 2s demonstrated that

Discussion

From a previous study, we identified that the ring finger protein-encoding gene, RNF36, is expressed during spermatid stage in semineferous tubules [1]. We suspected that RNF36 may play a role during spermiogenesis. The chromosome localization identified by FISH analysis showed that RNF36 is localized at the F1 region on mouse chromosome 2 that is equivalent to human chromosome 15q13. According to the database information from The National Center for Biotechnology Information, there has been no

Acknowledgements

We thank Dr. K. Deen for his critical reading of manuscript. This work was supported in part by a research grant NSC-91-2311-B-001-084 from the National Science Council, Taiwan.

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